1. Technical Field
The present invention is related to a novel hybrid cell line. More particularly, the present invention is related to the production of a human-mouse hybrid cell line having monocyte-macrophage properties and capable of stably expressing attractant-specific biochemical reactions necessary for chemotaxis to certain peptide probes.
2. State of the Art
The use of advanced genetic and biochemical techniques has led to a fairly detailed understanding of the bacterial sensory mechanism. However, progress on the biochemical mechanism of mammalian cell chemotaxis has been limited primarily due to the unavailability of suitable experimental probes. The evaluation of the chemotactic behavior of cells treated with various compounds that result in decreased methylation is one approach that has been applied to both bacteria and mammalian cells. These studies indicate that methylation reactions may be involved in chemotaxis in both bacteria and mammalian cells.
The observation that N-formyl peptides are attractants for human neutrophils and monocytes suggested that this peptide may serve as a probe with which to study detailed binding of an attractant to a mammalian cell receptor. Several studies of the binding of N-formyl peptides to cells or membranes have indicated that the receptor may exist in more than one affinity state and that the binding can be affected by guanine nucleotides. These characteristics are reminiscent of the regulation of adenylate cyclase by guanine nucleotide binding proteins and are indicative that similar regulatory proteins may be involved in chemotaxis. The possible involvement of a guanine nucleotide binding protein in chemotaxis is supported by the observation that pertussis toxin is a potent inhibitor of macrophage chemotaxis.
Mouse macrophage cell line RAW264 has been used as a model system to evaluate biochemical reactions critical to mammalian cell chemotaxis. Inhibitor studies have shown that one or more methylation reactions are required for chemotaxis of RAW264 cells. A comparison of the metabolic alterations that occur upon treatment with the inhibitors and the effects produced on chemotactic activity led to the conclusion that methylation of phosphatidylethanolamine, previously thought to be involved in the transduction of the chemotactic signal, is not required for RAW264 chemotaxis.
The advantages of the RAW264 macrophage cell line as a model chemotaxis system relate to the ease with which the cells can be cultured and manipulated for chemotactic and biochemical assays and for the introduction of genetic changes. However, a limitation of the RAW264 cell line has been the lack of chemically defined attractants. The two attractants that have been described for RAW264 cells are endotoxin-activated mouse serum and lymphocyte-derived chemotaxis factor, a culture supernatant from mitogen-stimulated leukocytes. Both of these attractants are complex molecular mixtures with multiple biological activities. On the other hand, N-formylmethionine-leucine-phenylalanine (FMLP), a tripeptide attractant for leukocytes from humans and several animal species, is not an attractant for the mouse RAW264 cells. One way to overcome the problems imposed by the limited attractant specificity of the RAW264 cell line was to derive a cell line by fusion of thioguanine-resistant RAW264 cells with human leukocytes.